Na+- independent glucose transporters (GT's) represent a critical site of regulation for glucose entry into cells, and secondarily, regulation of the glucose derived cell osmolytes (osmotically active organic solutes), sorbitol and glycerophosphorylcholine (GPC) which increase with hyperosmotic stress. The goals of this project are: 1) cloning of the mouse HepG2 GT gene; 2) mutation of the HepG2 GT gene by homologous recombination (HR) in embryonic stem (ES) cells; 3) microinjection of HepG2 GT mutant ES cells into mouse blastocysts and the breeding of chimeric mice; 4) selection for chimerics containing the HepG2 GT mutation in their germ line, and breeding of heterozygous and homozygous HepG2 GT deficient mice; 5) study the role of this GT in cell osmoregulation of renal inner medulla and brain. The technology for creation of mice carrying a selective gene mutation is now available in Dr. Seidman's laboratory, Success of the project will provide two important functions: 1) Establishment of this valuable method in our institution, which can be applied to the study of other tranporter and non-transporter genes, and 2) availability of the GT deficient mouse for study of cell osmoregulation, including regulation of sorbitol and aldose reductase implicated in diabetic tissue complications.